Differential Effects of p38, MAPK, PI3K or Rho Kinase Inhibitors on Bacterial Phagocytosis and Efferocytosis by Macrophages in COPD

Pulmonary inflammation and bacterial colonization are central to the pathogenesis of chronic obstructive pulmonary disease (COPD). Defects in macrophage phagocytosis of both bacteria and apoptotic cells contribute to the COPD phenotype. Small molecule inhibitors with anti-inflammatory activity against p38 mitogen activated protein kinases (MAPKs), phosphatidyl-inositol-3 kinase (PI3K) and Rho kinase (ROCK) are being investigated as novel therapeutics in COPD. Concerns exist, however, about off-target effects. We investigated the effect of p38 MAPK inhibitors (VX745 and SCIO469), specific inhibitors of PI3K α (NVS-P13K-2), δ (NVS-P13K-3) or γ (NVS-P13K-5) and a ROCK inhibitor PF4950834 on macrophage phagocytosis, early intracellular killing of bacteria and efferocytosis of apoptotic neutrophils. Alveolar macrophages (AM) obtained from broncho-alveolar lavage (BAL) or monocyte-derived macrophages (MDM) from COPD patients (GOLD stage II/III) enrolled from a well characterized clinical cohort (MRC COPD-MAP consortium) or from healthy ex-smoker controls were studied. Both COPD AM and MDM exhibited lower levels of bacterial phagocytosis (using Streptococcus pneumoniae and non-typeable Haemophilus influenzae) and efferocytosis than healthy controls. None of the inhibitors altered bacterial internalization or early intracellular bacterial killing in AM or MDM. Conversely PF4950834, but not other inhibitors, enhanced efferocytosis in COPD AM and MDM. These results suggest none of these inhibitors are likely to exacerbate phagocytosis-related defects in COPD, while confirming ROCK inhibitors can enhance efferocytosis in COPD

Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress

► Nrf2 anti-oxidant function is impaired when HDAC activity is inhibited. ► HDAC inhibition decreases Nrf2 protein stability. ► HDAC2 is involved in reduced Nrf2 stability and both correlate in COPD samples. ► HDAC inhibition increases Nrf2 acetylation

p38, PI3K and ROCK inhibition modulates signalling in alveolar macrophages.

<p><b>(A-F)</b> COPD alveolar macrophages (AM) were pre-treated with the designated concentrations of SCIO469 (A) VX745 (B), NVS-PI3K-2/3/5 (C-E), or PF4950834 (F), were then challenged with <i>S</i>. <i>pneumoniae</i> for 6 h, before cells were lysed and probed for either p-HSP27 (A-B), p-AKT (C-E), or p-MLC (F). Plots are representative of three independent experiments and densitometry from all three experiments are shown, * = p<0.05, ** = p<0.01, ANOVA with Dunnetts post-test vs control.</p

COPD alveolar macrophages have reduced phagocytosis of <i>S</i>. <i>pneumoniae</i>, which is not modified by p38, PI3K or ROCK inhibition.

<p><b>(A)</b> Alveolar macrophages (AM) from COPD patients or healthy controls were challenged with <i>S</i>. <i>pneumoniae</i> (Spn) at a multiplicity of infection (MOI) of 10. 4 h post challenge, the number of viable intracellular bacteria was determined. Data presented as median ± IQR, n = 10/14 healthy/COPD, *** = p<0.001, Mann-Whitney U test. <b>(B-D)</b> Healthy or COPD AM were treated with vehicle (-) or the designated doses of SCIO469, VX745 (B), NVS-PI3K-2/3/5, (C) or PF4950834 (D) before challenge with Spn at MOI 10. 4 h post challenge, numbers of viable internalized bacteria were determined, n = 3–5, data shown as paired vehicle and compound data for each donor, ns = non-significant, paired t- test.</p

Demographics of participants in AM experiments.

<p>Demographics of participants in AM experiments.</p

COPD MDM have reduced phagocytosis of <i>S</i>. <i>pneumoniae</i> which is not modified by p38, PI3K or ROCK inhibition.

<p><b>(A-B)</b> MDM from healthy donors or patients with COPD were incubated with fluorescent beads (A) or fluorescently labelled <i>S</i>. <i>pneumoniae</i> (B) for 4h and phagocytosis measured by fluorimetry. Data are presented as individual data points and the line represents median *p<0.05 Mann-Whitney U test. <b>(C-D)</b> MDM from healthy donors or patients with COPD were pre-incubated with p38 inhibitors VX745 (C) or SCIO469 (B) for 1h prior to challenge with fluorescently labelled <i>S</i>. <i>pneumoniae</i> for 4h. Data are presented as mean ± SEM for n = 10 healthy donors and n = 6 COPD. <b>(E-G)</b> MDM from healthy donors or patients with COPD were pre-incubated with the PI3K inhibitors NVS-PI3-2 (E), NVS-PI3-3 (F) or NVS-PI3-5 (G) for 2h prior to challenge with fluorescently labelled <i>S</i>. <i>pneumoniae</i> for 4h. Data presented as mean ± SEM for n = 3 healthy and n = 3 COPD. <b>(H)</b> MDM from healthy donors or patients with COPD were pre-incubated for 2h with the ROCK inhibitor, PF4950834 prior to challenge with fluorescently labelled <i>S</i>. <i>pneumoniae</i> for 4h. Data are presented as mean ± SEM for n = 3 healthy and n = 3 COPD. In all experiments, no significant differences were observed in internalization.</p

Inhibition of ROCK, but not p38 or PI3K pathways, increases efferocytosis in COPD alveolar and monocyte-derived macrophages.

<p>Alveolar (AM) or monocyte-derived macrophages (MDM) were incubated with PKH-26 stained apoptotic neutrophils for 90 min, before efferocytosis was assessed by flow cytometry. (<b>A-B)</b> Pooled vehicle data for AM, (A) n = 4–9, *** = p<0.001, Mann-Whitney and MDM (B), n = 7–12, ** = p<0.01, Student t-test. <b>(C-H)</b> Healthy or COPD AM (C, E and G) were pre-treated with vehicle (-) or 1μM SCIO469, 1μM VX745 (C), 100nM NVS-PI3K-2/3/5 (E), or 200nM PF4950834 (G) (+). MDM (D, F and H) were treated with vehicle (-) or compounds at the designated dose (+), ns = non significant, * = p<0.05, Wilcoxon matched pairs test.</p

Cytotoxicity and efficacy of p38, PI3K and ROCK inhibition in macrophages.

<p><b>(A-F)</b> Alveolar macrophages (AM) from COPD patients or healthy controls were incubated with either vehicle (-), or incubated with (+) 1μM SCIO469, 1μM VX745, 100nM NVS-PI3K-2/3/5 or 200nM PF4950834 for 20 h, before cultures were assessed for apoptosis (A-C) by nuclear fragmentation, or necrosis (D-F). In all experiments, n = 4, there was no significant differences between groups.</p

p38, PI3K or ROCK inhibition does not affect early-phase bacterial killing in alveolar macrophages.

<p><b>(A)</b> Alveolar macrophages (AM) from healthy donors or COPD patients were challenged with <i>S</i>. <i>pneumoniae</i> (Spn) at a multiplicity of infection of 10. 2 h after challenge non-internalised bacteria were washed off, and antibiotics added. At the designated time post-antimicrobials persisting viable bacteria were measured. <b>(B-D)</b> COPD AM were pre-treated with vehicle or the designated inhibitor before being challenged with Spn at MOI of 10. At the designated time post-antimicrobials persisting viable bacteria were measured. In all experiments, n = 4, with no significant differences between any groups at any time point, Friedman test.</p

p38, PI3K and ROCK inhibition modulates cytokine production in alveolar macrophages.

<p><b>(A-F)</b> COPD alveolar macrophages (AM) were pre-treated with the designated concentrations of SCIO469 or VX745 (A and D), NVS-PI3K-2/3/5 (B and E), or PF4950834 (C and F), before challenge with <i>S</i>. <i>pneumoniae</i> for 6 h. Supernatants were collected and levels of TNFα (A-C) and IL-6 (D-F) were measured by ELISA, n = 4, * = p<0.05, ANOVA with Dunnetts post-test vs control.</p